We have shown that the gel filtration profile of 125I-T4 (T4) equilibrated with the total lipoprotein fraction from normal human plasma separates several HDL particles which bind T4. Most of the T4 is recovered in the smaller size HDL subfraction (corresponding to HDL3). We also have shown that T4 binding to HDL is mediated by apolipoproteins, especially apoA-I. We have continued to characterize T4 binding to HDL subclasses by photoaffinity labeling with 0.5nM T4. Four preparations of HDL2 and five of HDL3 were labeled in the presence or absence of excess T4 or inhibitors of T4 binding to plasma proteins. Labeled HDL was analyzed by SDS-PAGE and radioactivity associated with apoA-I quantitated. The results showed that HDL3-apoA-I binds T4 more avidly and more consistently than HDL2-apoA-I. Inhibition of T4 binding was also variable and was generally more effective with HDL2. This suggests that the apoA-I in the two major HDL subclasses exists in different conformations having different abilities to bind T4. In further studies we find that photoaffinity labeling (PAL) of intact HDL with T4 results in the labeling of several other apos including apo(a), apoB100, apoA-II, apoA-IV and apoE.